Expression Kinetics of Nucleoside-Modified mRNA Delivered with Lipid Nanoparticles and Effects of Delivery Route
2015 · paper · Biomedicine
Studies protein production from nucleoside-modified mRNA in lipid nanoparticles injected into mice by six routes. Expression lasted up to 10 days at local injection sites and 1–4 days in the liver after systemic delivery, indicating these nanoparticles can carry mRNA.
Checked: PDF from pmc.ncbi.nlm.nih.gov, fetched 5 Oct 2026. Page numbers are those of that PDF.
6 findings
Substance Results 3.1: "cells were exposed to Lipofectin-mRNA complexes for an hour in serum-free medium whereas cells were incubated with TransIT-mRNA and LNP-mRNA complexes in complete medium", but Methods 2.5 state "mRNA-LNPs were added to the cells cultured in 194 ul AIM V medium", not in the complete medium defined in 2.2.
- p. 5 Section 3.1, first paragraph
cells were incubated with TransIT-mRNA and LNP-mRNA complexes in complete medium
- p. 4 Section 2.5
mRNA-LNPs were added to the cells cultured in 194 μl AIM V medium
- p. 3 Section 2.2
10% fetal calf serum (FCS) (HyClone) (complete medium)
- p. 5 3.1
incubated with TransIT-mRNA and LNP-mRNA complexes in complete medium
Only the authors can say what is right here.
Judge's note: Read Results 3.1 on extract/png/main-p005.png (crops C007-a, C007-d) and Methods 2.5 on main-p004.png (crops C007-b, C007-e). Section 2.2 (read in extract/main.txt, page 3, crop C007-c) defines "complete medium" specifically as DMEM with 2 mM L-glutamine and 10% FCS. In the same Methods paragraph the TransIT condition is explicitly in "183 ul complete medium" while the LNP condition is in "194 ul AIM V medium", so the author's own terminology distinguishes the two. The Results sentence, which is making a point about the conditions under which the three reagents were compared in Fig. 1A, therefore contradicts the Methods for the LNP arm. Which of the two statements is correct is for the authors.
Second check: another model agreed
Report a problem with this finding
Substance Results 3.1 and the Discussion make dose-response claims about Fig. 1B - "Transfecting higher amounts of cationic polymer and/or lipid-complexed Luc mRNA resulted in higher protein levels at 12 and 24 hours post transfection (Fig. 1B)", "LNP-formulated mRNA translated most efficiently at 24 hours post transfection regardless of the amount of mRNA transfected into cells (Fig. 1B)" and "more mRNA-LNPs did not result in higher luciferase expression (Fig. 1B)" - but Fig. 1B shows a single mRNA amount per reagent (1, 0.3 and 0.1 ug over Lipofectin, TransIT and LNP), with three bars per group corresponding to the 6, 12 and 24 h timepoints.
- p. 6 Section 3.1, second paragraph
Transfecting higher amounts of cationic polymer and/or lipid-complexed Luc mRNA resulted in higher protein levels at 12 and 24 hours post transfection (Fig. 1B)
- p. 6 Section 3.1, second paragraph
regardless of the amount of mRNA transfected into cells (Fig. 1B)
- p. 9 Discussion, second paragraph
more mRNA-LNPs did not result in higher luciferase expression (Fig. 1B)
- p. 14 Fig. 1B panel header
μg mRNA 1 0.3 0.1
Only the authors can say what is right here.
Judge's note: Examined Fig. 1B on extract/png/main-p014.png: the header "ug mRNA" carries three dose labels, 1, 0.3 and 0.1, each centred over one of the three x-axis groups Lipofectin, TransIT and LNP; each group contains three bars matching the three-shade 6h/12h/24h key, and the legend (crops C011-f, C011-i) mentions only "6, 12, and 24 hours", no doses. There is thus no dose series for any single formulation in the panel, so it cannot show either an increasing-dose effect for the cationic reagents or the absence of one for LNPs. The one cross-reagent comparison available runs the other way: the 1 ug Lipofectin bars are far lower than the 0.3 ug TransIT bars. Text read on main-p006.png (crops C011-a, C011-b, C011-g, C011-j) and main-p009.png (crops C011-c, C011-e). Fig. 1sup is cited only for in vivo dose response and is not among the files, so whether a dose series exists elsewhere is for the authors.
Second check: another model agreed
Report a problem with this finding
Reference or label Methods 2.3: "mRNA was purified by Fast Protein Liquid Chromatography (FPLC) (Akta Purifier, GE Healthcare) as described earlier15", while the same purification step is called HPLC everywhere else - abstract "HPLC purified, 1-methylpseudouridine-containing mRNA", Introduction, Section 2.4 "HPLC-purified 1-methylpseudouridine-containing firefly luciferase-encoding mRNA", Discussion and Conclusion.
- p. 4 Section 2.3
mRNA was purified by Fast Protein Liquid Chromatography (FPLC) (Akta Purifier, GE Healthcare) as described earlier15
- p. 4 Section 2.4, second paragraph
HPLC-purified 1-methylpseudouridine-containing firefly luciferase-encoding mRNA
- p. 1 Abstract
HPLC purified, 1-methylpseudouridine-containing mRNA
- p. 4 2.3
mRNA was purified by Fast Protein Liquid Chromatography (FPLC)
Only the authors can say what is right here.
Judge's note: Read Section 2.3 and 2.4 on extract/png/main-p004.png (crops C006-a, C006-b, C006-d, C006-e), the abstract on main-p001.png (crops C006-c, C006-f), and the Discussion/Conclusion wording on main-p008.png and main-p010.png ("HPLC-purification and incorporation of modified nucleosides", "nucleoside-modified and HPLC-purified mRNA"). The only description of the purification itself names FPLC and expands the acronym in full; the cited reference 15 is titled "HPLC purification of in vitro transcribed long RNA". The paper never states that the two labels denote the same procedure, so the technique attached to the material is inconsistently named within the article. The data are unaffected; it is the label that conflicts.
Second check: another model agreed
Report a problem with this finding
Wording The duration of intramuscular expression at the injection site is given two ways: "significant bioluminescent signal could be measured in the lungs and muscles, as well, with the latter lasting for up to 8 days post injection" (3.2) and "in the muscle could be measured for up to 8 days ... (Fig. 3 and Fig. 4)" (Discussion), versus "In both cases, translation levels decreased gradually at a much slower rate over time and the duration of protein production was extended, up to 10 days post injection" for intramuscular and intradermal delivery (3.3) and "intramuscular and intradermal injection ... translated locally at the site of injection for up to 10 days" (Abstract).
- p. 6 Section 3.2, second paragraph
with the latter lasting for up to 8 days post injection
- p. 7 Section 3.3, third paragraph
In both cases, translation levels decreased gradually at a much slower rate over time and the duration of protein production was extended, up to 10 days post injection
- p. 10 Discussion
in the muscle could be measured for up to 8 days
- p. 1 Abstract
Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10 days
Only the authors can say what is right here.
Judge's note: Read 3.2 on extract/png/main-p006.png (crop C012-a), 3.3 on main-p007.png (crops C012-b, C012-f, C012-g), the Discussion on main-p010.png (crops C012-c, C012-e, C012-i) and the abstract on main-p001.png (crop C012-d). In Fig. 2 (main-p015.png) the i.m. column of images ends at day 8 (only the i.d. column continues, to day 9), which fits the 8-day statements; in Fig. 3C (main-p016.png, crop C012-h) the x-axis runs to day 10 and the i.m. curve continues well past day 8. The paper nowhere distinguishes the two readings, and the Discussion attaches the 8-day figure to Fig. 3 and Fig. 4 rather than to the Fig. 2 image series, so the same quantity is reported with two different values and the ambiguity is left unresolved; only the authors can say which applies.
Second check: another model agreed
Report a problem with this finding
Wording Section 2.4 gives the TransIT complex as made up "in a final volume of 18 ul DMEM, which was added to a single 96-well of cells", while Section 2.5 states "17 ul of complex was added to cells cultured in 183 ul complete medium" - two values for the same added volume.
- p. 4 Section 2.4
in a final volume of 18 μl DMEM, which was added to a single 96-well of cells
- p. 4 Section 2.5
17 μl of complex was added to cells cultured in 183 μl complete medium
Only the authors can say what is right here.
Judge's note: Both sentences read on the page image extract/png/main-p004.png (crops C005-a and C005-b). Section 2.4 states the 18 ul final volume was itself added to the well; Section 2.5 states 17 ul was added, which with 183 ul of medium makes a round 200 ul well volume (18 ul would make 201 ul). The paper gives no dead-volume or pipetting allowance that would reconcile the two numbers, so the volume actually delivered per well is left ambiguous; only the authors can say which figure is right.
Second check: another model agreed
Report a problem with this finding
Wording "very low doses (0.005 mg/kg) of mRNA-LNPs could be translated for several days following the tested delivery routes" is unqualified, but Results 3.3 says of the intratracheal route "No signal could be measured when mice were injected with 0.1 μg of mRNA-LNPs" and Fig. 3A shows the i.v. curve only at 0.2 and 1 day.
- p. 9 Discussion, third paragraph
very low doses (0.005 mg/kg) of mRNA-LNPs could be translated for several days following the tested delivery routes
- p. 7 Section 3.3, first paragraph
No signal could be measured when mice were injected with 0.1 μg of mRNA-LNPs.
- p. 16 Fig. 3A
0.1 μg/injection
- p. 9 Discussion, 3rd paragraph (page 9 of the article)
Importantly, very low doses (0.005 mg/kg) of mRNA-LNPs could be translated for several days following the tested delivery routes
Only the authors can say what is right here.
Judge's note: The Introduction equates 0.1 μg with 0.005 mg/kg. Page 7 image: the no-signal sentence closes the intratracheal passage. Page 16 image, Fig. 3A (0.1 μg/injection): no i.t. curve; the i.v. (red) curve has points at 0.2 and 1 day only; i.p., i.m., s.c. and i.d. run to days 4-5. Page 17 image, Fig. 4: no 0.1 μg bar for i.t. So the statement holds for four of the six routes and not, as worded, for i.t. and i.v. I found no qualifying statement elsewhere in the text. This is overgeneralised wording; the authors should say which routes are meant.
Second check: another model agreed
Report a problem with this finding
9 minor (typos and the like; most checked by the judge only)
Reference or label Introduction: "Incorporation of modified nucleosides, such as pseudouridine or 1-methylpseudouridine and HPLC purification of the in vitro transcribed mRNA further increase protein translation and make mRNA immunologically silent2" — the HPLC-purification half of the claim is supported only by reference 2, which the reference list gives as the pseudouridine-incorporation paper.
- p. 2 Introduction
HPLC purification of the in vitro transcribed mRNA further increase protein translation and make mRNA immunologically silent2
Only the authors can say what is right here.
Judge's note: Read the sentence and its superscript 2 on crop C004-a (page 2), and the reference list in extract/main.txt / pages 11-12. Reference 2 is "Kariko K, et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Molecular therapy 2008; 16:1833-1840" - its title concerns nucleoside modification, not chromatographic purification. The paper's own HPLC-purification source is reference 18, "Kariko K, Muramatsu H, Ludwig J, Weissman D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic acids research 2011", which is cited elsewhere (Results 3.1, Discussion) but not here. The citation as printed is therefore incomplete for the HPLC clause; whether to add ref 18 is for the authors.
Second check: another model agreed
Report a problem with this finding
Reference or label "ionizable amino lipids of the type described by Jayaraman et al. has enabled the clinical development of siRNA therapeutics for hepatic gene silencing9,10": Jayaraman et al. is reference 7, but the sentence carries no citation to it; the only citations (9,10) are Akinc 2008 and Love 2010.
- p. 8 Discussion, second paragraph (continues on PDF page 9)
ionizable amino lipids of the type described by Jayaraman et al. has enabled the clinical development of siRNA therapeutics for hepatic
- p. 9 Discussion, top line
gene silencing9,10
- p. 12 Reference list
7. Jayaraman M, et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing
- p. 8 Discussion
described by Jayaraman et al. has enabled the clinical development of siRNA therapeutics for hepatic
Judge's note: Pages 8-9 images: the sentence names Jayaraman et al. with no superscript and ends with superscript 9,10. Page 12 image: ref 7 is Jayaraman M, et al.; ref 9 is Akinc A, et al. 2008; ref 10 is Love KT, et al. 2010. Refs 9,10 plausibly support the gene-silencing clause (the Introduction uses them the same way), so they are not necessarily wrong, but the named work is left uncited where it is named. Evident fix: add ref 7 after "Jayaraman et al.". Minor.
Second check: another model agreed
Report a problem with this finding
Wording Discussion p.10: "Intraperitoneal, intravenous, and deep intramuscular injections of mRNA-LNPs resulted in strong protein production in the liver. The duration of protein expression in the liver differed from other tissues in that it was short ... i.e., a short half-life of translation (Table 1)" — but Table 1 lists one half-life per delivery site, 14.8 h for intraperitoneal and 20.6 h for intramuscular, the second-longest in the table, so the cited table does not show a short half-life for two of the three routes named.
- p. 1 Abstract
resulting in active translation of the mRNA in the liver for 1–4 days
- p. 6 Section 3.2, first paragraph
there was no measurable luciferase activity at 3 days after mRNA-LNP injection
- p. 10 Discussion
return to baseline at day 3 post-injection (Fig. 2)
- p. 10 Discussion, page 10, paragraph 1
Intraperitoneal, intravenous, and deep intramuscular injections of mRNA-LNPs resulted in strong protein production in the liver. The duration of protein expression in the liver differed from other tissues in that it was short with a rapid decrease in luciferase enzyme activity and return to baseline at day 3 post-injection (Fig. 2), i.e., a short half-life of translation (Table 1).
Only the authors can say what is right here.
Judge's note: Read the Discussion sentence on extract/png/main-p010.png and crops C001-c/C001-d, and Table 1 on extract/png/main-p018.png (crops C001-e/C001-f). Table 1 as printed: intradermal 29.6, intramuscular 20.6, subcutaneous 14.7, intravenous 6.8, intraperitoneal 14.8, intratracheal 7.5 h. Of the three routes named in the sentence, only intravenous (6.8 h) is short; intramuscular (20.6 h) is the second longest value in the table and intraperitoneal (14.8 h) is longer than subcutaneous. Results 3.4 (extract/png/main-p008.png) itself attributes the shortest half-life only to "intravenous and lung". Neither Table 1, its heading ("Half-life of protein production") nor any footnote says the tabulated value is liver-specific for i.p./i.m., so the pointer does not support the claim it is attached to; only the authors can say whether the table was meant to be read tissue-by-tissue. The abstract "1-4 days" part of this cluster is adjudicated under C003.
Second check: another model agreed
Report a problem with this finding
Wording Abstract: "resulting in active translation of the mRNA in the liver for 1-4 days", while Results 3.2 state "there was no measurable luciferase activity at 3 days after mRNA-LNP injection" in the liver and "the majority of translation ceasing at day 2 post-injection", and the Discussion states "return to baseline at day 3 post-injection (Fig. 2)".
- p. 1 Abstract
resulting in active translation of the mRNA in the liver for 1–4 days
- p. 10 Discussion, page 10, paragraph 1
return to baseline at day 3 post-injection (Fig. 2)
- p. 6 Results 3.2
there was no measurable luciferase activity at 3 days after mRNA-LNP injection
- p. 6 Results 3.2, second paragraph
the majority of translation ceasing at day 2 post-injection
Only the authors can say what is right here.
Judge's note: Read the abstract on extract/png/main-p001.png (crop C003-a), Results 3.2 on main-p006.png (crops C003-c, C003-d) and the Discussion on main-p010.png (crop C003-b). No place in the article reports liver signal beyond day 3: for i.v./i.p. the liver signal is reported absent at day 3, for i.m./i.t. the majority of liver translation ceases at day 2, and the longer-lasting signal is explicitly attributed to the injection site (eyes, abdomen) rather than the liver. Fig. 2 (main-p015.png) and Fig. 3 (main-p016.png) are whole-animal images/total flux and are not resolved by organ, so they cannot supply a 4-day liver value either. The upper bound of the abstract's range is therefore unsupported by the body as printed; the abstract does not say which route or dose it refers to, so only the authors can resolve it.
Second check: another model agreed
Report a problem with this finding
Wording Results 3.3, on intravenous delivery: "Two days after the mRNA-LNP delivery, translation levels were 2 orders of magnitude lower compared to those measured at 4 hours and baseline expression was observed at day 3 post transfection", without saying that this refers to the liver only, whereas the cited Fig. 3C plots total flux and its i.v. curve has points at days 3, 4 and 5 clearly above the 2.E+05 axis floor.
- p. 7 3.3
baseline expression was observed at day 3 post transfection
- p. 16 Fig. 3C
i.v.
Only the authors can say what is right here.
Judge's note: Read 3.3 on extract/png/main-p007.png (crop C015-a) and Fig. 3C on main-p016.png (crop C015-b). The article does supply a possible reconciliation elsewhere - 3.2 (main-p006.png) says liver activity was gone by day 3 while "a small fraction of activity remained at the site of the injections (around the eyes and in the abdomen)" for up to 7 days, and Fig. 4's legend repeats the eye-socket point - but 3.3 attaches its statement to Fig. 3, which is not resolved by organ, so as printed the sentence does not say which signal reaches baseline at day 3. On the same panel the i.v. peak at 0.2 d and the day-2 value are separated by closer to three decades than two, but these are values read off a small five-decade log plot rather than printed numbers, so I do not confirm that part on its own.
Second check: another model agreed
Report a problem with this finding
Typo Section 2.8 prints the trapezoid formula with unbalanced parentheses: "AUC=S t(i+1) - t(i))/2 X (C(i) + C(i+1)," - a closing parenthesis with no opening one and an opening parenthesis that is never closed.
- p. 5 Section 2.8
AUC=Σ ti+1 − ti)/2 X (Ci + Ci+1,
- p. 5 2.8
AUC=Σ ti+1 − ti)/2 X (Ci + Ci+1
Judge's note: Read on the page image extract/png/main-p005.png and on the enlarged crop C008-a, which shows "Area under the curve calculations were performed using the equation: AUC=S t_{i+1} - t_i)/2 X (C_i + C_{i+1}, where t_i is the starting time point". The missing "(" before t_{i+1} and the missing ")" after C_{i+1} are present in the typeset page, so this is not an extraction artefact. The intended trapezoidal formula is unambiguous from the surrounding definitions, so the defect is typographic.
Second check: none (judge only)
Report a problem with this finding
Typo Section 2.7: "using the Living IMAGE Software provided by Caliper" - the imaging software's name is set as "Living IMAGE".
- p. 5 2.7
Living IMAGE Software provided by Caliper
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: the IVIS analysis software sold by Caliper Life Sciences is named "Living Image", not "Living IMAGE". I verified on extract/png/main-p005.png and the enlarged crop C009-a that the page does print "Living IMAGE Software provided by Caliper", with IMAGE in full capitals, so the quotation is accurate; the correct form of the product name cannot be checked from the files, and the issue is purely one of capitalisation.
Second check: none (judge only)
Report a problem with this finding
Typo Number-agreement slips: "To fulfill the first criteria" (should be "criterion"/"criteria" without "first") and "high amounts of protein was made" (should be "were made").
- p. 8 Discussion para 1
To fulfill the first criteria
- p. 10 Discussion
high amounts of protein was made
Judge's note: Both phrases read as printed on the page 8 and page 10 images. Grammar only; meaning is unaffected. The file is an unedited author manuscript.
Second check: none (judge only)
Report a problem with this finding
Typo Fig. 4 legend: "this amount was a substantial fraction delivered for the 0.1 μg dose" appears to be missing words (e.g. "fraction of the amount delivered").
- p. 17 Fig. 4 legend
this amount was a substantial fraction delivered for the 0.1 μg dose
Judge's note: Page 17 image shows the sentence as quoted. It is awkward or ungrammatical as printed, though the meaning is recoverable from the parallel sentence in Results 3.3. Cosmetic only, in an unedited author manuscript.
Second check: none (judge only)